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JCB: ARTICLE

© The Rockefeller University Press $8.00The Journal of Cell Biology, Vol. 175, No. 6, December 18, 2006 993–1003http://www.jcb.org/cgi/doi/10.1083/jcb.200605114

JCB 993

IntroductionThe β4 integrin is a laminin receptor that mediates the stable

adhesion of epithelial cells to the basal membrane (when incor-

porated in hemidesmosomes) and movement of carcinoma cells

through the extracellular matrix (when redistributed at actin-

rich protrusions; Mercurio and Rabinovitz, 2001; Litjens et al.,

2006). These roles of β4 in modulating static or dynamic adhe-

sion and in translating positional cues into different cellular

fates are consistent with a conventional mechanochemical ac-

tivity that has been traditionally ascribed to integrins. But, in

addition to these properties, β4 is also endowed with an unorth-

odox function: it can be a positive regulator of the anchorage-

independent growth of neoplastic cells (Zahir et al., 2003;

Bertotti et al., 2005; Lipscomb et al., 2005). This function is

counterintuitive given the established notion that transformed

cells are able to grow and survive in the absence of adhesion

and, thus, without demanding integrin-derived signals (Guo and

Giancotti, 2004). Nonetheless, this atypical behavior could ex-

plain the frequently observed up-regulation of β4 in human car-

cinomas (Mercurio and Rabinovitz, 2001) and suggests that β4

overexpression could afford cells with a selective advantage for

proliferation and survival in neoplastic contexts where tissue

architecture and canonical cell–matrix interactions are compro-

mised (Wilhelmsen et al., 2006).

A few modes of action have been proposed to explain this

feature. In some instances, carcinoma cells may secrete abun-

dant quantities of laminin-5, which can ligate β4 and sustain the

expansion of nonadherent colonies through activation of a Rac–

nuclear factor κB antiapoptotic pathway (Zahir et al., 2003).

Alternatively, the expression of β4 is accompanied by the trans-

lational up-regulation of VEGF, which can act as a survival fac-

tor favoring colony formation (Lipscomb et al., 2005). Finally,

we have recently demonstrated that β4 can promote anchorage-

independent growth in response to activation of the Met recep-

tor for hepatocyte growth factor (HGF; Bertotti et al., 2005).

This activity does not rely on laminin engagement, as it is

retained by a β4 truncated variant devoid of most of the extra-

cellular domain and requires Met-dependent tyrosine phosphor-

ylation of the β4 cytoplasmic domain. However, the signaling

pathway regulating this laminin-independent activity remains to

be elucidated. Using biochemical, pharmacological, and genetic

approaches, we found that the Met-mediated phosphorylation

of β4 initiates a previously unexplored transduction pathway

that involves Shp2 recruitment, Src activation, and Gab1 phos-

phorylation and ultimately leads to dedicated stimulation of the

β4 integrin activates a Shp2–Src signaling pathway that sustains HGF-induced anchorage-independent growth

Andrea Bertotti, Paolo M. Comoglio, and Livio Trusolino

Division of Molecular Oncology, Institute for Cancer Research and Treatment, University of Torino School of Medicine, 10060 Candiolo (Torino), Italy

Despite being a cell–matrix adhesion molecule, β4

integrin can prompt the multiplication of neoplastic

cells dislodged from their substrates (anchorage-

independent growth). However, the molecular events under-

lying this atypical behavior remain partly unexplored.

We found that activation of the Met receptor for hepato-

cyte growth factor results in the tyrosine phosphoryla-

tion of β4, which is instrumental for integrin-mediated

recruitment of the tyrosine phosphatase Shp2. Shp2 bind-

ing to β4 enhances the activation of Src, which, in turn,

phosphorylates the multiadaptor Gab1 predominantly on

consensus sites for Grb2 association, leading to privileged

stimulation of the Ras–extracellular signal-regulated kinase

(ERK) cascade. This signaling axis can be inhibited by

small interfering RNA–mediated β4 depletion, by a β4

mutant unable to bind Shp2, and by pharmacological

and genetic inhibition of Shp2 or Src. Preservation of the

β4 docking sites for Shp2 as well as the integrity of Shp2,

Src, or ERK activity are required for the β4-mediated in-

duction of anchorage-independent growth. These results

unravel a novel pathway whereby β4 directs tyrosine

kinase–based signals toward adhesion-unrelated outcomes.

Correspondence to Livio Trusolino: livio.trusolino@ircc.it

Abbreviations used in this paper: ERK, extracellular signal-regulated kinase; HGF, hepatocyte growth factor.

JCB • VOLUME 175 • NUMBER 6 • 2006 994

MAPK/extracellular signal-regulated kinase (ERK) cascade.

These results substantiate the crucial role of β4 in epithelial tu-

morigenesis and describe a novel integrin-dependent signaling

pathway that could be exploited by growth factor receptors for

the implementation of oncogenic responses.

Results𝛃4 integrin recruits the tyrosine phosphatase Shp2We previously demonstrated that the β4 cytodomain can be

tyrosine phosphorylated by Met. This event potentiates HGF-

mediated cellular invasion through the stimulation of Ras and

PI3K (Trusolino et al., 2001) and enhances anchorage-independent

growth through still uncharacterized mechanisms (Bertotti et al.,

2005). Although the signals implicated in Met/β4-dependent

cell invasion also play a crucial role in cellular proliferation and

are likely to contribute to β4-mediated anchorage-independent

growth, the involvement of additional transduction pathways

cannot be excluded.

To begin to explore new signaling circuits potentially con-

trolled by β4 activity, we performed a progressive tyrosine/

phenylalanine mutagenesis along the β4 tail and mapped three

tyrosines (Tyr1257, Tyr1440, and Tyr1494) that, when sequen-

tially mutated into phenylalanines, cause a gradual reduction in

β4 phosphotyrosine content up to complete abrogation (Bertotti

et al., 2005). Interestingly, an in silico analysis of the sequences

surrounding these tyrosines revealed common structural fea-

tures (Fig. 1 A): Tyr1257 and Tyr1494 are both located within

immune T cell inhibitory motifs that have been characterized as

canonical binding regions for protein and lipid phosphatases,

including the SH2-containing tyrosine phosphatase Shp2; simi-

larly, Tyr1440 is embedded in a degenerated consensus for

Shp2. Based on these observations, we hypothesized that

Tyr1257, Tyr1440, and Tyr1494 might be collectively involved

in central signaling functions whose trait of union is multiple

binding for Shp2.

To investigate this issue, we initially tested the potential

association between β4 and Shp2 using an overexpression system

(Fig. 1, B, C, and E). COS-7 cells were transiently transfected

with the following: (1) the wild-type forms of Met and β4,

a condition in which Met overexpression results in elevated

tyrosine phosphorylation of the integrin; (2) wild-type β4 and

a kinase-inactive variant of Met (MetK−), which is unable to

phosphorylate the integrin; (3) wild-type Met and β4 mutants

bearing single (β4-∆Shp2S), double (β4-∆Shp2D), or triple

(β4-∆Shp2T) phenylalanine substitutions of the three critical

tyrosines, with the consequent progressive reduction of β4 tyro-

sine phosphorylation levels (Fig. 1, B and D); and (4) wild-type

β4 alone or wild-type Met alone as negative controls.

First, we examined direct interaction in vitro between β4

and Shp2 in far-Western analysis using a GST fusion protein

of the Shp2 N-terminal SH2-containing domain (GST-Shp2).

This probe recognized β4 immunoprecipitates when the integrin

was fully phosphorylated by activated Met but not when Met-

dependent phosphorylation was inhibited (Fig. 1 B). Association

between GST-Shp2 and β4 was gradually weaker when using

the single and double β4 mutants and was totally prevented

upon expression of the β4 triple mutant (Fig. 1 B), indicating

that all of the putative consensus sites for Shp2 are involved

in this interaction. Notably, the effi cacy of Shp2 binding paral-

leled the extent of β4 phosphorylation, suggesting that the

tyrosines involved in Shp2 recruitment contain the bulk of

the integrin substrate capacity for Met (Fig. 1 B). Tyrosine

phosphorylation of β4 as well as Shp2 binding could not be ob-

served in the absence of transfected Met (Fig. 1 B). Similarly,

the transfection of Met alone was ineffective because COS-7

cells do not express endogenous α6β4 (Niessen et al., 1997).

When Tyr1257 and Tyr1494 were mutated into phenylalanines,

the overall phosphotyrosine content of β4 partially decreased,

and β4–Shp2 interaction was less effi cient; however, the com-

plete abolition of β4 phosphorylation and Shp2 association was

obtained only upon the phenylalanine permutation of Tyr1440

(Fig. 1 B). Thus, in principle, Tyr1440 could autonomously

account for all of the observed binding events. To rule out this

possibility, we repeated the far-Western experiment using a β4

mutant bearing a single phenylalanine substitution of Tyr1440

(β4-Y1440F). In COS-7 cells overexpressing Met and β4-

Y1440F, the tyrosine phosphorylation of β4 and interaction be-

tween β4 and Shp2 were reduced but not abolished (Fig. 1 C).

This indicates that Tyr1440 is necessary but not suffi cient for

Shp2 association and that Tyr1257 and Tyr1494 display a resid-

ual binding activity for the phosphatase.

Next, we analyzed association in vivo by coimmunopre-

cipitation experiments in COS-7 cells overexpressing Shp2 and

the various forms of Met and β4. Met has been reported to as-

sociate with both Shp2 (Fixman et al., 1996) and β4 (Trusolino

et al., 2001). Therefore, it may act as a bridging molecule be-

tween the integrin and the phosphatase. To exclude this possi-

bility and assess a direct interaction between β4 and Shp2, we

exploited a Met variant (MetD) that retains full catalytic activity

(therefore, it is still able to phosphorylate β4) but contains phe-

nylalanine substitutions of Tyr1349 and Tyr1356, which re-

present the docking residues for several signal transducers,

including Shp2 (Ponzetto et al., 1994; Fixman et al., 1996;

Furge et al., 2000). This mutant was validated for its inability to

bind Shp2 (Fig. 1 D) and for its capacity to phosphorylate β4

(Fig. 1 E) upon transient transfection in COS-7 cells. Under

these conditions, Shp2 could be effi ciently recovered in β4 im-

munoprecipitates. Conversely, the interaction was abolished in

the absence of β4 phosphorylation or in the presence of the β4

triple mutant displaying complete substitution of the critical

tyrosines and was progressively diminished in the single and

double mutants (Fig. 1 E). Again, the effi ciency of the β4–Shp2

interaction paralleled the extent of β4 phosphorylation; no tyro-

sine phosphorylation of β4 nor association with Shp2 could be

detected when β4 or Met were transfected individually (Fig. 1 E).

Finally, we assessed whether tyrosine-phosphorylated β4

can also recruit Shp2 in cells expressing endogenous proteins.

Accordingly, we set up coimmunoprecipitation experiments in

GTL16 cells, which express a constitutively active form of Met,

and in FG2 cells, in which Met phosphorylation is inducible by

HGF. Indeed, the association of Shp2 with β4 could be detected

basally in GTL16 (Fig. 1 F) and only after ligand stimulation in

β4 INTEGRIN STIMULATES SHP2–SRC SIGNALING • BERTOTTI ET AL. 995

Figure 1. Tyrosine-phosphorylated 𝛃4 interacts with Shp2. (A) Schematic representation of β4 structure. The cytoplasmic domain contains two pairs of type III fi bronectin-like repeats (FNIII) separated by a connecting segment (CS). The consensus sequences for Shp2 and tyrosine positions are indicated. TM, transmembrane domain. (B) β4–Shp2 association by far-Western analysis. COS-7 cells were cotransfected with wild-type (WT) or kinase-dead (K−) Met and with wild-type β4 or β4 mutants bearing single (β4-∆Shp2S; Y1257F), double (β4-∆Shp2D; Y1257F and Y1494F), and triple (β4-∆Shp2T; Y1257F, Y1440F, and Y1494F) phenylalanine substitutions of the potential docking tyrosines for Shp2. Lysates from each experimental point were subdivided into two aliquots and immunoprecipitated (IP) in parallel with antibodies to β4; Western blots (WB) were probed with antiphosphotyrosine (P-Tyr) antibodies (left) or with a GST-Shp2 fusion protein followed by anti-GST antibodies (right). The blots were then reprobed with anti-β4 antibodies. A fraction of the lysates (1/50) was loaded to detect Met from total extracts. In COS-7 cells, exogenous Met is expressed as both the precursor (top band) and mature form (bottom band). The weak band observable in the sixth lane corresponds to endogenous Met. (C) A β4 mutant with a single phenylalanine permutation of Tyr1440 (β4-Y1440F) displays reduced but detectable tyrosine phosphorylation and can bind Shp2 in far-Western analysis. (D) Coimmunoprecipitation experiment showing that wild-type Met but not a Met mutant with phenylalanine substitutions of the two tyrosines of the multidocking site (MetD) associates with Shp2. (E) Coimmunoprecipitation of β4 and Shp2 from lysates of COS-7 cells overexpressing Shp2 and the indicated forms of Met and β4. (F and G) Coimmunoprecipitation of β4 and Shp2 in cells with the endogenous expression of both proteins. (F) Reciprocal coimmunoprecipitation experiment in GTL16 cells. Cell lysates were immunoprecipitated with antibodies to myc (negative control), Shp2 (positive control), or β4. Blots were probed with anti-bodies against Shp2 (top), β4 (middle), or phosphotyrosine (P-Tyr; bottom). Shp2 is present in β4 immunoprecipitates (top), and β4 is present in Shp2 immunoprecipitates (middle). Immunopurifi ed β4 and Shp2-associated β4 appear to be constitutively tyrosine phosphorylated. (G) FG2 cells were either left untreated or were treated with 100 ng/ml HGF for 10 min. Lysates were immunoprecipitated with antibodies to myc or β4, and blots were probed as indicated. Shp2 can be recovered from β4 immunoprecipitates after HGF stimulation (top). Treatment with HGF induces the tyrosine phosphorylation of β4 (bottom).

JCB • VOLUME 175 • NUMBER 6 • 2006 996

FG2 cells (Fig. 1 G). The binding between tyrosine- phosphorylated

β4 and Shp2 is specifi c, as no coimmunoprecipitation of Shp2

was observed with antibodies against an unrelated myc antigen

(Fig. 1, F and G). As expected, β4 tyrosine phosphorylation was

constitutive in GTL16 cells and was induced by HGF in FG2

cells (Fig. 1, F and G).

Association between 𝛃4 and Shp2 leads to increased phosphorylation of Gab1 through a Src-dependent mechanismTranslocation of Shp2 at the plasma membrane is essential for

its function (Neel et al., 2003); therefore, the interaction of Shp2

with the intracellular domain of β4 is likely to stimulate Shp2-

dependent transduction pathways. One of the best-documented

activities of Shp2 relies on its ability to stimulate Src catalysis

by controlling Csk recruitment (Ren et al., 2004; Zhang et al.,

2004). This notion, together with the established observation

that Src can promote anchorage-independent growth when ab-

errantly activated (Yeatman, 2004), prompted us to investigate

whether the signaling contribution of β4 to this process could

entail the stimulation of Src.

To explore this subject, we examined the activation of Src

in MDA-MB-435 breast carcinoma cells, which do express Met

but are devoid of β4, and their counterpart cells stably trans-

fected with a human β4 cDNA (MDA-MB-435–β4 cells; Fig.

2 A). Under basal conditions, Src enzymatic function was already

higher in β4 cells compared with mock cells, possibly as a re-

sult of residual Met-based β4 signaling or to adhesion-triggered

Src stimulation as previously demonstrated (Dans et al., 2001;

Gagnoux-Palacios et al., 2003). This priming favored the fur-

ther activation of Src in response to HGF; indeed, ligand stimu-

lation led to a rapid boost of Src activity in β4 cells, whereas the

HGF-dependent activation of Src in mock cells displayed slower

kinetics and weaker intensity (Fig. 2 B). To evaluate whether

the stronger and accelerated activation of Src in β4-expressing

cells is caused by the integrin ability to bind Shp2, we generated

MDA-MB-435 transfectants expressing the β4 triple variant

that is unable to bind Shp2 (hereafter referred to as β4∆Shp2).

This mutant was exposed at the cell surface as effi ciently as

wild-type β4 together with the endogenous α6 subunit (Fig. 2 A).

In line with our hypothesis, disruption of the association be-

tween β4 and Shp2 almost abolished the HGF-dependent acti-

vation of Src (Fig. 2 B). Interestingly, the β4∆Shp2 mutant affected

the stimulation of Src more potently than the simple absence

of β4, suggesting that this variant could not only behave as

a signaling-dead molecule but also as a dominant repressor

of other endogenous β4 signaling partners. Accordingly, β4

has been shown to impinge on signals emanating from the

Ron tyrosine kinase receptor (Santoro et al., 2003), the EGF

receptor (Mainiero et al., 1996; Rabinovitz et al., 1999), Erb-B2

(Gambaletta et al., 2000; Guo et al., 2006), and tetraspanins

(Yang et al., 2004).

Next, we searched for a Src substrate that could be a po-

tential target of this Met–β4–Shp2 signaling pathway. The Gab1

multiadaptor protein is the major Met substrate (Birchmeier

et al., 2003) and can also be phosphorylated by Src (Chan et al.,

2003). To verify whether the activation of Src in β4-expressing

cells supports a more effi cient phosphorylation of Gab1 in

response to HGF, we analyzed Gab1 phosphorylation levels upon

HGF stimulation in mock and β4 cells. In time-course experi-

ments, the HGF-induced phosphorylation of Gab1 was more

intense and durable in β4 than in mock cells (Fig. 2 C). The in-

creased phosphorylation of Gab1 in β4 cells likely depends on

Shp2 recruitment; indeed, the HGF treatment of cells express-

ing the β4∆Shp2 mutant led to a less effi cient phosphorylation of

Gab1 compared with cells expressing wild-type β4 (Fig. 2 C).

Likewise, stimulation with increasing amounts of HGF pro-

duced a weak dose-dependent curve of Gab1 phosphorylation

in mock cells. Conversely, Gab1 phosphorylation in β4 cells

reached a plateau at a very low concentration of ligand, and

the overall phosphorylation levels were more robust than in

mock transfectants (Fig. 2 D). Again, the response of β4∆Shp2

cells was similar or even lower than that showed by mock

cells (Fig. 2 D).

In a complementary approach, we abated β4 levels by len-

tiviral delivery of siRNA in MDA-MB-231 breast carcinoma

cells, which endogenously synthesize both Met and the integrin

(Fig. 2 E). We then reestablished β4 expression in β4-defi cient

cells using a siRNA-resistant variant that lacks most of the ex-

tracellular portion, including the siRNA target region, but re-

tains the ability to transduce Met-dependent responses (β4∆extra;

Trusolino et al., 2001; Bertotti et al., 2005). In addition to wild-

type β4∆extra, which displays an intact cytoplasmic domain, we

also expressed a β4∆extra mutant with phenylalanine substitu-

tions of the tyrosines involved in Shp2 binding (Fig. 2 E). In all

cases, Met expression was unaffected (Fig. 2 E). Consistent with

that observed in MDA-MB-435 cells, β4 knockdown in MDA-

MB-231 resulted in the reduced HGF-dependent phosphorylation

of Gab1, whereas the rescue of β4 expression was accompa-

nied by the restoration of higher Gab1 phosphorylation levels

in the presence of a signaling-competent cytoplasmic portion

but not when Shp2 recruitment was abolished (Fig. 2 F).

If Shp2 and Src specifi cally contribute to Gab1 phospho-

tyrosine content in a β4-dependent manner, the attenuation of

Shp2 and/or Src activity should affect the HGF-triggered phos-

phorylation of Gab1 only in cells expressing β4. In fact, Shp2

inhibition by expression of a catalytically inactive dominant

interfering isoform (Shp2DN) resulted in the decreased phos-

phorylation of Gab1 in MDA-MB-435–β4 cells but not in mock

cells (Fig. 2 G). Similar results were obtained when mock and

β4 cells were treated with the Src pharmacological inhibitor

PP2 (Fig. 2 H) or upon transfection of a kinase-dead variant of

Src (SrcDN; Fig. 2 I).

𝛃4-dependent activation of Src leads to the selective association of Gab1 with Grb2Gab1 is a scaffolding adaptor that associates with a variety

of signal transducers after phosphorylation on multiple sites

(Gu and Neel, 2003). We wondered whether the Src-dependent

increase of Gab1 phosphorylation in β4 cells leads to a generic,

quantitative enhancement of the overall Gab1 phosphotyro-

sine content or to a selective, qualitative phosphorylation of

defi ned tyrosine residues with consequent specifi c binding to

a given transducer.

β4 INTEGRIN STIMULATES SHP2–SRC SIGNALING • BERTOTTI ET AL. 997

Figure 2. Association between 𝛃4 and Shp2 leads to the increased phosphorylation of Gab1 through a Src-dependent mechanism. (A) Expression of β4 or the β4 mutant unable to bind Shp2 (β4∆Shp2) in MDA-MB-435 cells. Both molecules are exposed at the cell surface, as assessed by surface biotinylation (top), and are expressed at comparable levels, as shown in total cell extracts (middle). Equal loading was verifi ed by probing the blot with antiactin antibod-ies (bottom). Surface biotinylation detects the entire α6β4 integrin complex. (B) In vitro kinase assay measuring Src activation upon stimulation with 100 ng/ml HGF at different time points in mock (blue), β4 (green), and β4∆Shp2 (red) MDA-MB-435 cells. Data are the means ± SEM (error bars) of fi ve inde pendent experiments performed in duplicate. (C) Differential Gab1 phosphorylation upon HGF stimulation for different times in mock, β4, and β4∆Shp2 cells. MDA-MB-435 transfectants were stimulated with 50 ng/ml HGF, and lysates were immunoprecipitated and probed as indicated. Dotted lines indicate the grouping of images from different parts of the same gel. Gab1 phosphorylation was quantitated by densitometric analysis (OD) relative to Gab1 protein content in immunoprecipitates. (D) Differential Gab1 phosphorylation upon stimulation with different doses of HGF for 30 min in mock, β4, and β4∆Shp2 cells. (E) Western blot analysis showing β4 expression in lysates from MDA-MB-231 cells infected with a scrambled (ctr) siRNA or with a β4-specifi c siRNA; β4 siRNA cells were reinfected with wild-type β4∆extra or with a ∆Shp2 mutant. In all transfectants, Met expression was unaffected. (F) Differential Gab1 phosphorylation in the different MDA-MB-231 transfectants. (G–I) Inhibition of Shp2 and Src impairs the HGF-dependent phosphorylation of Gab1 in MDA-MB-435–β4 but not in mock cells. Cells were transiently transfected with a control vector (empty) or with catalytically inactive isoforms of either Shp2 (Shp2DN; G) or Src (SrcDN; I).The exogenous expression of Shp2 and Src is shown in the bottom panels (G and I). In H, cells were pretreated with 5 μM PP2 for 30 min. The PP2-mediated inhibition of Src was assessed with an antibody against active Src (bottom; H). HGF was given at 50 ng/ml for 30 min.

JCB • VOLUME 175 • NUMBER 6 • 2006 998

To investigate this issue, we overexpressed Gab1 in the

various MDA-MB-435 transfectants and assessed the ability of

Gab1 to form supramolecular complexes with a representative

panel of SH2-containing signaling molecules, including Grb2,

Shc, PI3K, and Shp2 itself. Among these signaling effectors,

Grb2 appeared to bind Gab1 more effi ciently in β4 versus mock

cells after HGF stimulation (Fig. 3 A). This binding was re-

duced in cells expressing the β4∆Shp2 mutant (Fig. 3 B); simi-

larly, the PP2-mediated inhibition of Src, which decreases

HGF-induced Gab1 phosphorylation only in β4 cells, impaired

the association of Grb2 with Gab1 in the presence but not in the

absence of β4 (Fig. 3 B).

Grb2 can bind tyrosine-phosphorylated Gab1 using the

SH2 domain, but it also combines with Gab1 in a phosphorylation-

independent manner using the SH3 domain (Holgado-Madruga

et al., 1996). To verify that the increased association between

Gab1 and Grb2 in β4 cells is in fact caused by the enhanced

HGF-dependent phosphorylation of the Gab1 tyrosines respon-

sible for SH2-mediated Grb2 binding, we performed a far-

Western analysis using a GST fusion protein of the Grb2 SH2

domain (GST-SH2-Grb2). In accordance with the coimmuno-

precipitation data, the interaction between GST-SH2-Grb2 and

Gab1 after HGF stimulation was much more effi cient in β4

cells. Again, binding was decreased in cells expressing the

β4∆Shp2 mutant, and treatment with PP2 impaired Gab1–Grb2

association in β4 transfectants but not in mock cells (Fig. 3 C).

Together, these results indicate that the Shp2-mediated activa-

tion of Src in β4 cells leads to the dedicated phosphorylation

of Gab1 on tyrosine residues specifi cally responsible for Grb2

binding, a preferential association that can be abrogated by un-

coupling β4 from Shp2 or by hindering Src function. Privileged

interaction between Gab1 and Grb2 in β4 cells results in the

increased stimulation of Ras-dependent effectors: indeed, the

amplitude and persistence of ERK/MAPK activation after HGF

stimulation were higher in β4 compared with mock and β4∆Shp2

transfectants (Fig. 3 D).

Shp2 and Src are required to promote 𝛃4-mediated anchorage-independent growthTo verify whether the aforementioned signaling pathway is in

fact responsible for the ability of β4 to promote anchorage-

independent growth, we performed soft agar assays on several

different MDA-MB-435 transfectants (Figs. 2 E and 4 A). As

expected, the ectopic expression of β4 considerably enhanced

the basal and HGF-stimulated formation of suspended colonies

compared with mock cells both in absolute numbers (Fig. 4 B,

graph) and in size (Fig. 4 B, images). Analogous results were

obtained upon transfection of the β4∆extra variant, further vali-

dating the role of this nonadhesive mutant as an effi cient substi-

tute of wild-type β4 in HGF-driven responses and confi rming

the notion that the Met-dependent signaling activity of β4 does

not require laminin ligation (Fig. 4 B; Trusolino et al., 2001;

Bertotti et al., 2005). In contrast, expression of the β4∆Shp2

signaling-dead mutant was less effective, indicating that the

Figure 3. 𝛃4-dependent activation of Src leads to the selective association of Gab1 with Grb2 and to the increased activation of ERK/MAPKs. (A) HGF stimulation results in the in-creased association of Gab1 with Grb2 in β4 cells. MDA-MB-435 mock and β4 cells were transiently transfected with Gab1 and stimu-lated with 100 ng/ml HGF for 30 min. Lysates were immunoprecipitated with antibodies to Gab1, and blots were probed as indicated. Densitometric analysis of the blots obtained in four experiments indicates that the HGF- dependent association of Grb2 with Gab1 in mock cells is increased 1.26-fold (± 0.48) compared with nonstimulated cells, whereas the HGF-dependent association of Gab1 with Grb2 in β4 cells is increased 4.7-fold (± 0.98; P < 0.01). (B) Abrogation of Shp2 binding or Src inhibition with PP2 impairs the coimmuno-precipitation of Gab1 with Grb2 in β4 cells. Dotted lines indicate the grouping of images from different parts of the same gel. (C) Far-Western analysis showing the association between immunoprecipitated Gab1 and GST-SH2-Grb2. In accordance with the coimmunoprecipitation data, HGF-dependent Gab1–Grb2 interaction is increased in β4 cells compared with mock and β4∆Shp2 transfectants. PP2-mediated inhibi-tion of Src results in decreased Gab1–Grb2 in-teraction in β4 but not in mock cells. (D) HGF activates ERK/MAPKs longer and more effi -ciently in β4 cells than in mock or β4∆Shp2 cells. Cells were treated with 100 ng/ml HGF for the indicated times, and total lysates were probed on blots as indicated. Band intensities were quantitated by densitometric analysis with respect to total protein content.

β4 INTEGRIN STIMULATES SHP2–SRC SIGNALING • BERTOTTI ET AL. 999

interaction between β4 and Shp2 is critical for this process

(Fig. 4 B). Transfection of dominant-negative isoforms of

Shp2 or Src in β4-expressing cells (Fig. 4 B) as well as

treatment of β4 cells with the Src inhibitor PP2 or with the

ERK inhibitor PD98059 (Fig. 4 C) potently impaired HGF-

dependent clonogenic activity in soft agar. Together, these

results reinforce the observation that Shp2, Src, and ERKs

are central downstream transducers of β4-driven anchorage-

independent growth.

In a complementary way, siRNA-mediated reduction of

β4 expression in MDA-MB-231 cells led to an almost complete

abolition of basal and HGF-stimulated anchorage-independent

growth. Colony-forming ability was partially reestablished

upon the expression of the β4∆extra siRNA-resistant variant dis-

playing a wild-type intracellular domain but not upon expres-

sion of the ∆Shp2 mutant (Fig. 5).

DiscussionWhen a normal cell of epithelial origin happens to lose contact

with the basement membrane, the surrounding stromal com-

partment impedes its multiplication and, in the meantime, stim-

ulates its apoptotic elimination (Frisch and Screaton, 2001).

To avoid this and to ensure successful metastatic dissemination,

malignant elements undergo an adaptive process that relies

mainly on two strategies. One is the abnormal and unpolarized

secretion of basement membrane molecules, which aberrantly

reconstitute the original histological niche, thus imparting sur-

rogate proliferative and survival signals. Parallel (or alternative)

to this is the hyperactivation of oncogenic pathways, which pro-

duce a global amplifi cation of the cell signaling activity with

consequent elusion of the external adhesive consensus (Hanahan

and Weinberg, 2000). Whatever the mechanism used, the

Figure 4. 𝛃4 sustains HGF-induced anchorage-independent growth through the activation of Shp2, Src, and ERK. (A) Western blot analy-sis showing expression of the indicated cDNAs in MDA-MB-435 cells subjected to soft agar assays. Filters probed with anti-Shp2 and -Src antibodies were deliberately underexposed to better visualize the overexpression of exog-enous proteins. (B) Representative images and quantitation of soft agar assays in MDA-MB-435 cells with ectopic expression of the indicated proteins in the absence (NS) or presence of 30 ng/ml HGF. Pictures show col-ony aspect at high magnifi cation, whereas the graph reports colony numbers as counted at low magnifi cation over the entire microscopic fi eld. Light bars, no stimulation; dark bars, HGF. (C) Representative images and quantita-tion of soft agar assays in MDA-MB-435 cells expressing β4 that were either left untreated (NS) or were treated with HGF in the absence or presence of the Src inhibitor PP2 or the ERK inhibitor PD98059. Data are the means ± SEM (error bars) of two independent experiments performed in triplicate. **, P < 0.01 with respect to all other transfectants. Bars, 400 μm.

JCB • VOLUME 175 • NUMBER 6 • 2006 1000

phenotypic outcome of this process is that neoplastic cells ac-

quire the ability to grow in the absence of proper anchorage.

In this study, we show that the β4 integrin conspires with

the tyrosine kinase Met for effi cient execution of anchorage-

independent growth by channeling Met signals toward activa-

tion of the Ras-ERK oncogenic cascade. This function relies on

Met-triggered phosphorylation of the β4 cytoplasmic domain

and on the ensuing recruitment of the tyrosine phosphatase

Shp2 to the integrin tail. β4–Shp2 association results in the

stimulation of Src, which, in turn, favors a more effi cient phos-

phorylation of the Gab1 multiadaptor protein, mainly on tyro-

sines involved in Grb2 binding. Ultimately, privileged interaction

between Gab1 and Grb2 leads to dedicated stimulation of the

MAPK–ERK pathway.

Structural aspects of 𝛃4–Shp2 interactionAssociation between β4 and Shp2 depends on the integrity of

three tyrosines along the β4 cytoplasmic domain (Tyr1257,

Tyr1440, and Tyr1494) that are inserted in canonical or degene-

rated motifs for the SH2 domain of Shp2 and, consequently,

could act as docking sites for the phosphatase when phosphory-

lated. However, based on the crystal structure of a fragment of

the β4 intracellular portion encompassing the fi rst pair of type

III fi bronectin-like (FNIII) repeats (de Pereda et al., 1999), in-

teraction between Shp2 and Tyr1257 appears to be more com-

plicated because this tyrosine is part of the hydrophobic core of

the second repeat (Fig. 1 A). Thus, it may not be easily accessi-

ble for phosphorylation because of conformational constraints.

This information is in disagreement with our fi ndings and

with a previous study showing the phosphorylation of Tyr1257

in response to the antibody-mediated ligation of β4 (Shaw,

2001). A possible explanation for this discrepancy is that al-

though the aromatic ring of Tyr1257 is in fact involved in hy-

drophobic interactions with other residues and is not accessible

to the solvent, the side chain is not completely buried in the

FNIII core so that the OH group reaches the solvent-accessible

surface of the protein. This kind of spatial organization seems

to be permissive for phosphorylation; for example, the human

Vaccinia H1-related phosphatase is phosphorylated by ZAP-70

at Tyr138 despite that only the OH group of the amino acid is

partially exposed to the solvent (Alonso et al., 2003). Moreover,

physical association with Met could affect the structural organi-

zation of β4 and induce a more relaxed conformation of the

FNIII repeat, facilitating recognition by the kinase and transfer

of the phosphate group.

In any case, our observation that the phenylalanine per-

mutation of Tyr1257 results in reduction of the overall β4 phos-

photyrosine content and in a less effi cient association with Shp2

does not exclude the possibility that Tyr1257 is not a direct

phosphorylation site, but it is necessary for the phosphoryla-

tion of other residues that, in turn, could be involved in Shp2

recruitment. This hypothesis is in agreement with the general

notion that bindings between SH2 domains and the correspond-

ing ligand motifs hardly occur in rigid structural environments

such as the FNIII fold.

A novel signaling pathway connecting 𝛃4 integrin to ERK activation: biochemical and biological insightsThe importance of the signaling axis explored here in β4-

mediated anchorage-independent growth is underscored by the ob-

servations that a β4 mutant unable to bind Shp2 cannot sustain

this process and that interference with Shp2, Src, or ERK im-

pairs the biological activity of wild-type β4. Interestingly, Shp2

is known to act downstream from Met when physically associ-

ated with Gab1 to sustain the prolonged stimulation of ERKs

(Maroun et al., 2000). However, the signaling intermediates pro-

pagating this activity remain elusive (Gu and Neel, 2003). Our

fi ndings confi rm the crucial role of Shp2 as a positive regulator

of Ras-dependent signals in response to HGF and provide an al-

ternative pathway as well as several additional molecular actors

that connect Met function to the enhancement of ERK activity.

The unorthodox observation that β4 contributes to the

malignant phenotype by acting as a biochemical selector of tu-

morigenic signaling pathways unveils an unforeseen function

that transcends the activity of β4 as an integrin and assimilates

it to classical signal relay adapters endowed with oncogenic

Figure 5. 𝛃4 sustains HGF-induced anchorage-independent growth through the recruitment of Shp2. Representative images and quantitation of soft agar assays in MDA-MB-231 cells displaying the artifi cial manipula-tion of β4 levels as shown in Fig. 2 E. Data are the means ± SEM (error bars) of two independent experiments performed in triplicate. **, P < 0.01 with respect to all other transfectants. WT, wild type. Bar, 400 μm.

β4 INTEGRIN STIMULATES SHP2–SRC SIGNALING • BERTOTTI ET AL. 1001

potential. Something similar has been described for β1A integ-

rin, the most widely expressed spliced isoform of β1 integrins:

in this case, interaction between β1A and the insulin-like growth

factor-I tyrosine kinase receptor results in integrin-mediated re-

cruitment of the downstream effector IRS1, which inhibits cell

adhesion and stimulates insulin-like growth factor–dependent

cellular proliferation and tumor growth (Goel et al., 2004).

Most of the signaling effectors along the transduction path-

way discussed in this study are involved in human malignancies

in vivo: Met, β4, and Src are often overexpressed (and coex-

pressed) in a vast number of human carcinomas (Mercurio and

Rabinovitz, 2001; Trusolino and Comoglio, 2002; Ishizawar

and Parsons, 2004), and Shp2 gain of function mutations have

been described in some solid tumors and in various forms of

leukemia (Bentires-Alj et al., 2004). Gab2, a close homologue

of Gab1, is amplifi ed and overexpressed in a fraction of human

breast tumors and collaborates with the ErbB2 oncogene for

activation of a Shp2–ERK pathway that drives mammary carci-

nogenesis (Bentires-Alj et al., 2006). Intriguingly, β4 has re-

cently been shown to cooperate with ErbB2 in breast cancer

onset (Guo et al., 2006). Finally, constitutively active Src tran-

scriptionally up-regulates HGF in breast carcinoma cells, gen-

erating an autocrine loop that promotes cancer progression

(Wojcik et al., 2006). Altogether, these fi ndings suggest not

only a functional role in vivo but also a cooperative effect and a

potential epistatic relationship for these signals.

Finally, it is worth noting that the tyrosines involved in

Shp2 binding represent biochemical hotspots for β4 signaling

capacity also with respect to other transduction pathways:

Tyr1257 is necessary for β4 phosphorylation in response to

laminin ligation (Shaw, 2001), Tyr1440 is responsible for SH2-

mediated interaction with the Grb2 upstream effector Shc (Dans

et al., 2001), and Tyr1494 is involved in the adhesion-driven

stimulation of PI3K (Shaw, 2001). It will be interesting to ana-

lyze whether these additional transducers can complement or

substitute for the signaling axis identifi ed in this study for fos-

tering β4-mediated anchorage-independent growth and possi-

bly tumorigenesis.

Met and 𝛃4 in cancer onset and progressionAccumulating evidence suggests that the aptitude of a neoplasm

to disseminate at distant sites is already preordained by the pro-

tein repertoire that progenitor cells express relatively early in

tumorigenesis. This indicates that the phenotype acquired en

route by an evolving tumor cell can confer not only a selective

advantage for cell replication (a condition necessary for forma-

tion of the primary tumor mass) but also, later during neoplas-

tic progression, the tendency to metastasize (Bernards and

Weinberg, 2002).

This line of thinking fi ts nicely with our fi ndings on the

role of β4 and Met in tumor evolution. We and others have dem-

onstrated that both molecules, individually or in combination,

are able to enhance the motility and survival of cancer cells as a

prelude for neoplastic invasion and metastasis (Mercurio and

Rabinovitz, 2001; Trusolino and Comoglio, 2002). Here, we

show that the same molecules can also cooperate for anchorage-

independent growth, which is the experimental and phenotypic

hallmark of primary tumorigenesis. Together, these data allow

us to draw a linear fl owchart along which β4 and Met are caus-

ally involved fi rst in the derailment of cell accretion and later in

the promotion of metastatic spread.

This dual activity of β4 and Met on epithelial cells is par-

alleled by their common ability to induce a peculiar phase of the

angiogenic process in endothelial cells. Specifi cally, both the

inhibition of β4 signaling by an intracellularly truncated β4

mutant (Nikolopoulos et al., 2004) and the inhibition of Met

function by a decoy receptor (Michieli et al., 2004) prevent

branching of medium- and small-size vessels into microvessels

during intratumor vascularization, suggesting that both mole-

cules are selectively involved in the onset of the invasive step of

pathological angiogenesis. At present, we do not know whether

this vascular phenocopy is supported by a functional collabora-

tion between β4 and Met. However, all of these observations

highlight the versatility of the β4–Met system in promoting the

various and sequential aspects of neoplastic progression.

Materials and methodsMaterials, antibodies, and vectorsWe used the following reagents: anti–human Met, anti-Shp2, anti-Src, anti-actin, GST-Shp2, and GST-SH2-Grb2 (Santa Cruz Biotechnology, Inc.); anti-β4 integrin (BD Biosciences and Chemicon); anti-Gab1, anti-Shc, anti-PI3K, antiphosphotyrosine, and anti-GST (Upstate Biotechnology); anti-Grb2 (Transduction Laboratories); antiactive and total ERK/MAPK (Promega); antiactive Src (nonphospho-Tyr529; Biosource International); PP2 (Calbio-chem); wild-type Shp2 and catalytically inactive C/S Shp2 in pJ3H vector (obtained from B.G. Neel, Harvard Medical School, Boston, MA); and wild-type Src and kinase-dead K/R Src in pCMV5 vector (obtained from J. Brugge, Harvard Medical School). The β4∆Shp2 construct containing phe-nylalanine mutations of Y1257, Y1440, and Y1494 was generated by PCR amplifi cation of the BssHII–NotI fragment of a β4 template already containing the Y1257F and Y1494F substitutions (obtained from L.M. Shaw, Harvard Medical School). To create the β4 siRNA expression vector, oligonucleotides used by Chung et al. (2004) were annealed and ligated into pSUPER between the BglII and HindIII sites. BamHI- and XhoI-digested inserts were then subcloned into the pRLL5 lentiviral vector. A scrambled β4 oligonucleotide was used as a control. The constructs encoding for wild-type Met, kinase-inactive Met, wild-type β4, β4-1440F, and β4∆extra have been described previously (Trusolino et al., 2001). The β4 cDNA used in this study corresponds to PubMed accession no. AAC51632 and matches with the sequence originally cloned by Suzuki and Naitoh (1990).

Cell culture, transfection, and viral infectionCOS-7, MDA-MB-435, and MDA-MB-231 cells were cultured in DME supplemented with 10% FBS (Invitrogen). The expression of exogenous proteins was obtained with LipofectAMINE- or LipofectAMINE 2000 (Invitrogen)–mediated transfection according to the manufacturer’s proto-col or with retroviral or lentiviral infection. Viral hybrid vectors were pro-duced by the transient transfection of 293T cells. Viral supernatants were fi ltered through a 0.22-μm fi lter, and infections were performed in the pres-ence of 4 μg/ml polybrene (Sigma-Aldrich).

Biochemical methodsFor immunoprecipitations, 5 × 106 cells were lysed for 20 min at 4°C with 1 ml of a buffer containing 50 mM Hepes, pH 7.4, 5 mM EDTA, 2 mM EGTA, 150 mM NaCl, 10% glycerol, and 1% Triton X-100 in the presence of protease and phosphatase inhibitors. For β4–Shp2 coimmunoprecipita-tions in FG2 cells, 1% Brij58 was used instead of Triton X-100. Extracts were clarifi ed at 12,000 rpm for 15 min, normalized with the BCA Protein Assay Reagent kit (Pierce Chemical Co.), and incubated with different mAbs for 2 h at 4°C. Immune complexes were collected with either protein G– or protein A–Sepharose, washed in lysis buffer in the presence of 1 M LiCl, and eluted. Total cellular proteins were extracted by solubilizing the cells in boiling SDS buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1% SDS). Extracts were electrophoresed on SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Hybond; GE Healthcare).

JCB • VOLUME 175 • NUMBER 6 • 2006 1002

Nitrocellulose-bound antibodies were detected by the ECL system (GE Healthcare). Unless otherwise indicated, cells were stimulated with 50 ng/ml HGF for 30 min. PP2 was generally used at a 10-μM concentration and applied to cells for 30 min. In experiments aimed at analyzing the associa-tion of Gab1 with signal transducers and the HGF-dependent activation of ERKs, mock cells and cells expressing wild-type β4 or β4∆Shp2 were tran-siently transfected with a Gab1 cDNA.

For far-Western analysis, membranes were blocked for 1 h in TBS, 0.1% Tween-20, 5% BSA, and 3 μm glutathione and were incubated for 3 h with 1 μg/ml GST fusion protein previously adsorbed to 3 μM glutathione in 0.5% BSA. Filters were subsequently decorated with anti-GST antibody.

Src kinase assays were performed on Src immunoprecipitates using a commercial kit (Upstate Biotechnology) based on the phosphorylation of a specifi c substrate peptide (K V E K I G E G T Y G V V Y K ) using the transfer of the γ-phosphate of γ-[32P]ATP by Src. The phosphorylated substrate was then separated from the residual γ-[32P]ATP using phosphocellulose paper and quantifi ed with a scintillation counter.

Soft agar assay3,000 cells were resuspended in complete medium containing 0.5% Seaplaque agar. Cells were seeded in 24-well plates containing a 1% agar underlay and supplemented twice a week with complete medium. In some experiments, MDA-MB-435–β4 cells were treated twice a week with 5 μM PP2 or 10 μM PD98059. Colonies were stained by the incorporation of tetrazolium salts 2 (for MDA-MB-435) or 3 wk (for MDA-MB-231) after seeding. Colonies were coded and scored in a blinded fashion by a sec-ond observer. Colony numbers were obtained using a phase-contrast light microscope (DMIL; Leica) fi tted with a 32-grid eyepiece at a total magnifi -cation of 20×. Images were captured with ImageReady software (Adobe) using a microscope (DMIL; Leica) and a 20 × 0.30 objective (Leica) equipped with a digital camera (DFC320; Leica).

Statistical and densitometric analysisResults are means ± SEM. Comparisons were made using the two-tailed t test. P-values <0.05 were considered to be statistically signifi cant. Blot images were captured using a molecular imager (ChemiDoc XRS; Bio-Rad Laboratories). Densitometric analysis was performed with analysis software (Quantity One 1-D; Bio-Rad Laboratories) installed on the imager. Images were arranged and labeled using Illustrator (Adobe).

We are indebted to José M. de Pereda (University of Salamanca, Salamanca, Spain) for his helpful comments and discussion. We thank Leslie M. Shaw, Benjamin G. Neel, and Joan Brugge for reagents, Alessandra Crivellari and Francesco Galimi for help in some experiments, Raffaella Albano for technical assistance, Antonella Cignetto for secretarial assistance, and Catherine Tighe for editing the manuscript.

This work was supported by an Associazione Italiana per la Ricerca sul Cancro grant to P.M. Cornoglio and a Ministero dell’Istruzione dell’Università e della Ricerca (PRIN 2004) grant to L. Trusolino.

Submitted: 17 May 2006Accepted: 15 November 2006

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